The invention is generally directed to novel glycosylated steroid derivatives. These derivatives have a variety of uses, including but not limited to the general permeabilization of membranes, such as biological membranes (e.g., cellular, mucosal, gastrointestinal, blood-brain barrier, and the like). In particular, the present derivatives are useful in facilitating the transport of molecules across biological membranes. The facilitation is achieved by combining the present derivatives with the molecules of interest, either as a conjugate comprising the present derivative covalently linked directly or indirectly with the molecule of interest or as an admixture comprising the two main components. In this manner, the molecule of interest, especially those of a therapeutic significance (more, below) can better exhibit its activity, whether of a biological, physical or chemical nature. The invention is further directed to novel methods for the efficient synthesis of these derivatives, including their combinations with representative molecules of interest.
To elicit the desired biological response, a molecule of therapeutic significance, i.e., those having a diagnostic, prophylactic or therapeutic use (and termed herein "therapeutically-significant-molecule" or "therapeutically-significant-compound"), must be made available in an effective-concentration at its site of action. Many factors determine the concentration of a therapeutically-significant-compound, which ultimately reaches the site of action, including the amount administered, and the extent and rate of the compound's absorption, distribution, biotransformation, and excretion. (Goodman and Gilman, The Pharmacological Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., Inc., New York, 1980, pp. 1-39.) The foregoing factors may, in turn, be influenced by the chosen route of administration of the therapeutically-significant-compound.
The most common routes of administration of therapeutically-significant-compounds are parenteral (e.g., intravenous, subcutaneous, and intramuscular) and enteral (oral ingestion), although methods to administer therapeutically-significant-compounds across the skin (e.g., transdermal) or mucosa (e.g., oral, nasal, rectal, vaginal, and the like) also are known. Parenteral methods are considered to be extremely effective, in general, allowing for rapid increases in blood levels of a wide range of therapeutically-significant-compounds. Parenteral methods are advantageous in that they circumvent first-passage hepatic metabolism. However, parenteral administration of a therapeutically-significant-compound can cause pain, irritation, possible tissue damage over the long term, and carries a potential risk of infection. In addition, parenteral methods frequently are inconvenient, particularly those that are restricted to trained medical personnel (e.g., intravenous methods).
Enteral methods are more convenient than parenteral methods, and generally are more economical and acceptable to the recipients. However, orally administered, therapeutically-significant-compounds may be inefficiently absorbed (for example, they may decompose within the gastrointestinal tract or may simply pass through without absorption). Moreover, the time from ingestion to absorption may prohibit effective use in emergency situations. As stated above, certain therapeutically-significant-compounds cannot be orally administered as they are destroyed, prior to reaching their site of action, by the digestive enzymes, acid, and surface-active lipids in the gut. Other therapeutically-significant-compounds are subject to extensive, first-passage hepatic metabolism, rendering them ineffective following oral administration.
Non-parenteral methods which circumvent problems associated with instability of drug preparations in the gut and first-passage hepatic metabolism long have been sought. Administration via transdermal, oral mucosal, rectal, and nasal routes are among the alternatives which have been explored. Such alternatives further include administering the therapeutically-significant-compound orally, but encapsulated in a protective delivery system designed to extrude the contents at a predetermined point in the lower gastrointestinal tract. However, the efficacy of these alternative drug delivery methods often is limited by poor absorption of the therapeutically-significant-compounds at the site of delivery or application.
Effective strategies to enhance absorption of therapeutically-significant-molecules across membranes, such as mucosal membranes, cellular membranes, nuclear membranes, and the like, could enhance the efficacy of many known drug preparations that are poorly absorbed regardless of the method of administration. Such strategies to enhance trans-membrane absorption or penetration could be particularly useful for therapeutically-significant-compounds that are administered across the skin and mucosal tissues, including mucosal tissues of the gastrointestinal, genitourinary, and respiratory tracts.
The basic structural unit of biological membranes is a phospholipid bilayer, in which are embedded proteins of various size and composition. The surfaces of the phospholipid bilayer, which project into the aqueous cellular environment, are formed by the hydrophilic heads of the phospholipids; the interior of the bilayer is comprised of the fatty acyl hydrophobic tails. The membrane proteins may be involved in transport processes and also may serve as receptors in cellular regulatory mechanisms or signal transduction.
Natural mechanisms for traversal of biological membranes include passive diffusion, facilitated diffusion, active transport, receptor-mediated endocytosis and pinocytosis. Passive diffusion works best for small molecules that are lipid-soluble. However, biological membranes are essentially impermeable to most water-soluble molecules, such as nucleosides, amino acids, proteins, and other hydrophilic, therapeutically-significant-molecules. Such molecules enter cells via some type of carriermediated transport system in which specific entities facilitate traversal of the membrane. Natural carriers for facilitating traversal of the membrane are of limited utility, however, as such carriers will accept substrates of only a predetermined molecular configuration. Many therapeutically-significant-compounds are not efficiently absorbed because they are neither lipophilic enough to diffuse passively across cell membranes nor possess the structural features recognized by the natural transport systems.
Strategies to enhance the uptake of therapeutically-significant-molecules across biological membranes have been investigated previously and fall into two broad categories. The first category includes all strategies in which the structure of the therapeutically-significant-compound is changed, either by making the compound itself more lipophilic or by conjugating the compound to other entities known to interact with phospholipid membranes. The common goal has been to increase passive diffusion across the membrane by lowering the energy barrier to diffusion and/or by increasing the local concentration of the compound at the membrane surface.
As mentioned above, the first category includes the strategy of taking advantage of the cellular transport mechanism (either active or facilitated transport or receptor-mediated endocytosis) by conjugating the therapeutically-significant-compound to entities known to interact with the cellular transport machinery. Among the reported techniques to conjugate molecules of therapeutic significance to other entities is the work of Letsinger and others on oligonucleotidecholesterol conjugates. (See, Letsinger R. L. et al. "Cholesteryl-conjugated oligonucleotides: Synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture." Proc. Natl. Acad. Sci. USA (September 1989) 86:6553-6556; Stein C. A. et al. "Mode of Action of 5'-Linked Cholesteryl Phosphorothioate Oligodeoxynucleotides in Inhibiting Syncytia Formation and Infection by HIV-1 and HIV-2 in Vitro." Biochemistry (1991) 30:2439-2444.)
Targeting molecules to the brain requires traversal of the blood-brain barrier, a capillary including system with unique morphological characteristics, which acts as a system-wide cellular membrane separating the brain interstitial space from the blood. Like other biological membranes, the bloodbrain barrier is relatively impermeable to many hydrophilic, therapeutically-significant-compounds. The strategies which have been developed for targeting compounds to the brain include direct delivery by invasive procedures, intra-arterial infusion of hypertonic substances, and conversion of hydrophilic compounds to lipid-soluble entities.
U.S. Pat. No. 4,902,505 describes a recent attempt to facilitate transport by coupling a hydrophilic peptide of interest to a peptide carrier which itself is capable of traversing the barrier via receptor-mediated transcytosis.
The second broad category to enhance uptake includes those strategies in which the therapeutically-significant-compound is administered to specific body surfaces as an admixture with other molecules that are known to permeabilize membranes. For example, several investigators have attempted to mix insulin with adjuvants, such as bile salts, which might enhance nasal insulin absorption. (See, Hirai et al. Int. J. Pharmaceutics (1981) 9:165-184; Hirai et al. Diabetes (1978) 27:296-199; British Patent No. 1,527,506; U.S. Pat. No. 4,153,689; and Pontiroli et al. Br. Med. J. (1982) 284:303-386.) EP 0 444 778 describes the use of alkyl saccharides to enhance the penetration of topically applied drugs across mucus-covered epithelial tissues, in general, and the corneal epithelium, in particular. U.S. Pat. No. 4,865,848 to Cheng et al., issued Sep. 12, 1989, discloses the use of sucrose esters, particularly sucrose monolaurate, for enhancing the transdermal flux of transdermally-delivered drugs. U.S. Pat. No. 4,746,508 to Carey et al., issued May 24, 1988, reports the use of fusidic acid and cephalosporin derivatives to increase the permeability of human and animal body surfaces to drugs.
The glycosylated steroid derivatives of the present invention may be used effectively in a strategy for enhancing the uptake of a second compound through a particular membrane, including the two broad categories discussed above. Indeed, it has been discovered that the instant derivatives can interact with a wide variety of membranes, including biological phospholipid membranes, thereby possessing the potential to enhance the penetration of therapeutically-significant-compounds through such membranes.
Like some of the previously used adjuvants and "enhancers" (e.g., cholic acid and fusidic acid derivatives), the novel derivatives of the present invention are amphiphilic in a facial sense; that is, one side or face of the molecule is hydrophobic while the opposite side or face is hydrophilic. However, the novel derivatives of the present invention have structural features which differ significantly from those of the previously known "enhancers". That is, the instant derivatives are glycosylated on the hydrophilic face of the molecule in a manner that is not shared by any previously known, facially-amphiphilic steroid.
The present inventors have discovered that glycosylation on the hydrophilic surfaces significantly changes both the solubility properties of the steroids and the manner in which they associate. Many of the instant glycosylated steroids have been shown by the inventors to be more effective than the parent, nonglycosylated steroids in permeabilizing both artificial and biological membranes. The novel, glycosylated steroid derivatives of the present invention, therefore, have been found to increase the delivery of therapeutically-significant-compounds across a variety of membranes. The enhanced transport is facilitated by combining the instant derivatives with the therapeutically-significant-compounds, either as admixtures or as conjugates therewith.
Prior to the present invention, no method existed for efficiently synthesizing all of the glycosylated steroid derivatives of the present invention. Many glycosylation reactions using thioglycosides have been reported. (See, Ferrier R. J. et al. "A Potentially Versatile Synthesis of Glycosides," Carbohydrate Research (1973) 27:55-61; Garegg P. J. et al. "A reinvestigation of glycosidation reactions using 1-thioglycosides as glycosyl donors and thiophilic cations as promoters," Carbohydrate Research (1983) 116:162-5; Nicolaou K. C. et al. "A Mild and General Method for the Synthesis of O-Glycosides," J. Am. Chem. Soc. (1983) 105:2430-2434; Lonn H. "Synthesis of a tri- and a hepta-saccharide which contain .alpha.-L-fucopyranosyl groups and are part of the complex type of carbohydrate moiety of glycoproteins," Research (1985) 139:105-113; Andersson F. et al. "Synthesis of 1,2-cis-linked glycosides using dimethyl(methylthio) sulfonium triflate as promoter and thioglycosides as glycosyl donors," Tetrahedron Letters (1986) 3919-3922; Brown D. S. et al. "Preparation of cyclic ether acetals from 2-benzenesulphonyl derivatives: a new mild glycosidation procedure." Tetrahedron Letters (1988) 29/38:4873-4876; Ito Y. et al. "Benzeneselenenyl triflate as a promoter of thioglycosides: a new method for O-glycosylation using thioglycosides," Tetrahedron Letters (1988) 10614; Dasgupta F. et al. "Alkyl sulfonyl triflate as activator in the thioglycoside-mediated formation of .beta.-glycosidic linkages during oligosaccharide synthesis," Carbohydrate Research (1988) 177:c13-c17.) However, none of these reported methods teach the use of a glycosyl sulfoxide as a glycosylating agent.
Utilization of an activated glycosyl sulfoxide intermediate in a process for glycosylating steroids, has been reported previously by the inventors in an article that appeared in the J. Am. Chem. Soc. (1989) 111:6881-2, the entire contents of which are incorporated by reference herein. However, the reported method represents only preliminary results on the glycosylation of steroids of the Formula (I). More specifically, further experimentation in the series has revealed unique reaction conditions that are necessary to achieve the efficient and stereoselective synthesis of glycosylated compounds of the Formula (I). In particular, it has been discovered that the reaction solvent plays a critical role in the stereoselectivity of glycosylation. Using a non-polar, aprotic solvent increases selectivity for alpha (.alpha.) glycosidic bond formation while the use of a polar, aprotic solvent such as propionitrile increases selectivity for beta (.beta.) glycosidic bond formation.
The type of sulfoxide used in the glycosylation reaction also affects the outcome of the reaction. For example, it is vital to use the para-methoxy phenyl sulfoxide as the leaving group in the novel process described herein to obtain good yields of beta (.beta.) selectivity in the glycosidic bond formation. The yield of the glycosylation reaction yielding alpha (.alpha.) or beta (.beta.) glycosidic linkages also may be increased by using less than one equivalent of triflic anhydride in the glycosylation process.
Finally, the identity of the protecting groups present on the glycosyl donor also have an impact on the stereochemical course of the glycosylation reaction. When the protecting group used is pivaloyl, only beta (.beta.) glycosidic bonds are formed in the glycosylation process, regardless of whether an aprotic, non-polar solvent or an aprotic, polar solvent is used for the reaction. The above-recited factors taken together indicate that one skilled in the art could not have practiced the invention without the detailed further experimentation provided herein.